MECHANISM OF LENGTH DEPENDENT CONTRACTILE REGULATION

心肌和骨骼肌收缩的调节是由 多个分子过程的复杂相互作用。我们的目的是 确定肌节长度变化的机制(S) 影响骨骼肌和心肌的力量(斯塔林定律)。 已经提出了完全基于细丝的机制， 基于粗丝的机构，包括实质上的调制 交叉桥连接的作用。其他机制，而不是 仅以粗丝或细丝为基础，涉及改变的肌丝 晶格间距或电荷密度。辨别亲人 我们将利用这些不同机制的贡献(如果有的话) 新开发的(I)抑制或修饰肌动球蛋白的方法 相互作用(金属氟化物、巯基试剂、阳离子多肽、 GTP)和(Ii)在没有钙的情况下激活结皮纤维)。激活 没有钙将使我们能够直接将钙从CrosBridge中分离出来 对长度调节和激活的影响，以及由于 CA2与TNC以外的位点结合，如肌球蛋白轻链。 由于力在许多实验中都会被抑制，我们将使用 荧光标记TNC检测细丝水平的研究 并且还将监测纤维硬度以确定程度， 以及跨桥连接的强度。肌钙蛋白互换 骨骼肌和心肌之间的亚基将被用来确定 如果细丝调节蛋白的性质影响其长度 力-钙关系的依赖性和收缩动力学。 我们还将确定肌球蛋白特性在 确定力和动力学的激活和长度依赖关系。 实验将在带皮的骨骼肌和心肌上进行； 心肌力的长度依赖性更强，这表明 长度调控的潜在机制甚至更有效。 虽然没有一种单一的实验方法是完美的，但数据的收敛 从我们提议的实验中获得的，将使我们能够 对这一现象的根本机制(S)得出强有力的结论 高度非线性的、复杂的系统间的协同作用 肌肉蛋白质导致收缩活动的长度调节。

The regulation of contraction in cardiac and skeletal muscle results from the complex interplay of multiple molecular processes. Our purpose is to determine the mechanism(s) by which changes in sarcomere length affects force in skeletal and cardiac muscle (Starling's Law). Exclusively thin filament-based mechanisms have been proposed, as have thick filament-based mechanisms which include a substantial modulatory role for crossbridge attachment. Other mechanisms, which are not exclusively thick or thin filament based, involve altered myofilament lattice spacing or charge density. To distinguish the relative contribution, if any, of these various mechanisms we will take advantage of newly developed methods for (i) inhibiting or modifying actomyosin interactions (metallofluorides, sulfhydryl reagents, cationic peptides, GTP) and (ii) activating skinned fibers without Ca2+). Activation without Ca2+ will enable us to directly separate Ca2+ from crossbridge effects on length regulation and activation, as well as effects due to Ca2+ binding to sites other than TnC, such as at myosin light chains. Since force will be inhibited in many experiments, we will use fluorescently labeled TnC to estimate the level of thin filament activation and will also monitor fiber stiffness to determine the degree, as well as the strength of crossbridge attachment. Exchange of troponin subunits between skeletal and cardiac muscle will be done to determine if the properties of thin filament regulatory proteins affect the length dependence of force-calcium relations and the kinetics of contraction. We will also determine the role that myosin properties play in determining the activation and length dependence of force and kinetics. Experiments will be done with both skinned skeletal and cardiac muscle; the steeper length dependence of force in cardiac muscle suggests that the underlying mechanisms of length regulation are even more potent. While no single experimental approach is perfect, the convergence of data to be obtained from the experiments which we propose, will enable us to reach strong conclusions about the underlying mechanism(s) by which this highly nonlinear, complex system of cooperative interactions between muscle proteins results in the length regulation of contractile activity.